This grant application is divided into two major sections based on our studies on the total synthesis and biosynthesis of two unrelated families of natural products: (1) taxol; and (2) the paraherquamides, stephacidins A and B, asperparalines and congeners. I. Taxol. The biosynthesis of taxol from the cyclization of geranylgeranyl pyrophosphate (ggPP) via taxa-4(5),11(12)-diene, will be studied in collaboration with Prof. Rod Croteau's laboratory at Washington State University. The sequence of hydroxylation reactions from this simple diterpenoid comprising the core A/B/C ring system of taxol shall be elucidated. In collaboration with Prof. Croteau, we have established that the first committed step in taxol biosynthesis is the cyclization of geranylgeranyl pyrophosphate (ggPP) to taxa-4(5),11(12)-diene. Further, we have established that the first hydroxylation reaction on taxadiene on the pathway to taxol is hydroxylation at C-5 giving taxa-4(20),11(12)-diene-5alpha-ol and that this substance is further acetylated to taxa-4(20),11(12)-diene-5alpha-acetate. Our objectives are to continue to map the sequence of hydroxylations from the monooxygenated metabolite to 10-deacetylbaccatin III that has the fully oxygenated taxol core. To elucidate the regio- and stereochemistry of the second, third, fourth, and fifth hydroxylation reactions in taxol biosynthesis, we specifically propose to utilize synthetic taxadiene-polyols as substrates to probe the downstream hydroxylation reactions. We have prepared the following diols: (1) taxa-4(20),11(12)-diene-2alpha, 5alpha-diol, (2) taxa-4(20),11(12)-diene-5alpha,10beta-diol, and the synthesis of (3) taxa-4(20),11(12)- diene-5alpha,9alpha-beta-diol is currently in progress. Synthetic access to candidate taxadiene triols: (1) taxa-4(20),11(12)-diene-2alpha,5alpha,10beta- triol, and (2) taxa-4(20),11(12)-diene-5alpha,9alpha-beta,10beta-triol will be pursued and used in conjunction with clones that express biosynthetic taxol cytochrome P450 oxidases that the Croteau lab has assembled from Taxus sp. using a PCR homology-based approach. The main objectives of these studies are to elucidate and target the rate-limiting steps of taxol biosynthesis with the ultimate objective of manipulating taxol biosynthesis at the genetic level and significantly increasing the yield of taxol from stabilized cell cultures of Taxus sp. II. Stephacidins, Paraherquamides & Asperparalines. The goals of this program are to utilize the tools of total synthesis to fully elucidate the biosynthetic pathway from the primary amino acids (isoleucine, proline, and tryptophan) and mevalonate-derived isoprene moieties to the polycyclic indole alkaloids comprising the paraherquamides, stephacidins, avrainvillamide and the asperparalines. Provocative evidence has been elucidated in our laboratory indicating that this class of alkaloids, are constructed by a rare biosynthetic intramolecular [4+2] cycloaddition reaction. Through the use of total chemical synthesis of isotopically labeled intermediate metabolites, key features of the biosynthetic pathways to these complex secondary metabolites will be experimentally elucidated. In collaboration with Prof. David Sherman's laboratory at the University of Michigan, we plan to isolate and clone the biosynthetic gene clusters for paraherquamide and stephacidin biosynthesis.